Hypothyroid serum control

ABSTRACT

Described is the preparation of a hypothyroid serum control using normal serum and serum containing elevated levels of thyroxine binding globulin. The control serum exhibits hypothyroid values in clinical tests that measure triiodothyronine uptake and thyroxine concentration.

United States Patent [1 1 Turner et al.

[4 Nov. 25, 1975 HYPOTHYROID SERUM CONTROL [.75] Inventors: James E. Turner, Madison; Michael B. Kenoff, Hackettstown, both of 1 [73] Assignee: Warner-Lambert Company, Morris Plains, NJ.

[22] Filed: May 28, 1974 [21] Appl. No.: 473,463

[52] US. Cl 23/230 B; 252/408; 424/1 '[51] Int. Cl? G01N 33/16 [58] Field of Search 23/230 B; 424/1; 252/408 [56] References Cited UNITED STATES PATENTS 6/1970 Murty et. a]. 23/230 B 3,743,482 7/1973 Eisentraut 23/230 B 3,775,615 11/1973 Eisentraut 23/230 B 3,776,698 12/1973 Eisentraut ..23/23()B Primary Examiner-R. E. Serwin Attorney, Agent, or Firm-Albert H. Graddis; Frank S. Chow; George M. Yahwak [57] ABSTRACT Described is the preparation of a hypothyroid serum control using normal serum and serum containing elevated levels of thyroxine binding globulin. The control serum exhibits hypothyroid values in clinical tests that measure triiodothyronine uptake and thyroxine concentration.

11 Claims, No Drawings HYPOTHYROID SERUM CONTROL To diagnose thyroid disfunction and evaluate thyroid therapy, clinicians rely on tests which measure a pa- .tients serum triiodothyronine (T uptake level as well as tests which do quantitate the thyroxine (T concentration. T uptake levels are determined in serum using T uptake tests (e.g., Triosorb); serum T, levels are guantitated using tests which measure total serum T.

concentration (e.g. T radioimmunoassay, Tetrasorb).

Tests measuring total serum T are dependent upon the actual thyroid hormone concentration present in the serum, whereas tests which measure T uptake depend upon the thyroxine-binding protein (TBP) concentration in the serum as well as the concentration of thyroid hormone. Both tests are important in forming a clear understanding of the patients thyroid state.

Circulating T and T are bound to several constituents of the blood of which the thyroxine binding globulin (TBG) fraction contains the major binding sites which are fixed in number. The binding of T and T on the TBG molecule is one of competitive protein binding, that is, unbound T, will replace T and T. already on the molecule.

As there is only l-2 mg of TBG/100 ml normal serum, the binding sites, equivalent to approximately micrograms of T per 100 ml of normal serum, are readily saturated by a small increase of T concentration.

In hyperthyroidism, the binding sites on the TBG molecules are nearly saturated with T and T in hypothyroidism, the binding sites are highly unsaturated, resulting in an increased ability for T uptake by the TBG molecule in the serum.

The Triosorb assay and other similar tests for T uptake operate on the principle of competitive protein binding. Serum T and T are primarily bound to the binding sites on the TBG molecule. The number of unoccupied binding sites is determined by the addition of radioactive T (T to serum in the presence of an adsorbing agent. When T is added to serum, any excess not bound to the binding sites of the TBG molecule in the serum will be adsorbed onto the added agent. Resin sponges are the adsorbing agents for the Triosorb test. For example, in hyperthyroidism, most of the TBG binding sites are occupied by T and T and thus the added T:,* will not be taken up by the endogenous TBG molecules, but will be taken up by the test adsorbent. In

hypothyroidism, the reverse is true. Thus, the amount of radioactive T bound by the adsorbent directly reflects the thyroid state of the patient.

The tetrasorb assay for T is also based upon the principle of competitive protein binding. To determine the T concentration, T is extracted from the serum releasing it from its binding protein, TBG. The serum extract is then added to a solution containing a limited quantity of exogenous TBG to which is bound radioactive T, (T., A displacement reaction occurs in which the T in the extract displaces the-T from the exogenous TBG. This displaced Tfi is then adsorbed onto the resin sponge used as the adsorbing agent. As more T is added, more T is displaced from the TBG. The amount of T displaced from the TBG is therefore directly proportional to the amount of T present in the serum extract. in hyperthyroidism there is more T available in the serum'extract to displace.T.,* from exogenous TBG than in euthyroidism or hypothyroidism.

When normal levels of TBG are present in serum, these types of thyroid function tests reflect the actual state of the patient. When TBG and T., levels are elevated, as in pregnancy or following estrogen ingestion in the form of oral contraceptives, T uptake tests indicate the patient to be hypothyroid. However, tests measuring total T indicate an euthyroid or sometimes a hyperthyroid condition. Simply stated, pregnancy or estrogen ingestion results in an increase in the number of TBG molecules causing an increase in hormone binding sites with a concurrent rise in thyroid hormone levels. Thyroid function tests carried out on these sera at this time show an increased T uptake, indicative of hypothyroid function and an increased T, level indicative of hyperthyroid function. It is the object of this invention to describe a control serum prepared from these types of sera and normal serum that will serve as a hypothyroid control serum for T, tests as well as T uptake tests. By normal serum Applicants mean serum having normal T and T values and which may be human, beef, sheep, goat, or other animal serum. Only horse serum has been found to lack utility.

A serum is hyperthyroid by the tetrasorb and triosorb tests if the percentage of T uptake is greater than 35, and the T concentration is above 14.5 mcg per ml of serum. A serum is euthyroid if the percentage of T uptake is 25 to 35 and the T concentration is 5.3 14.5 mcg per 100 ml of serum. Hypothyroid serum has a T uptake of below 25 percent and less than 5 mcg of T per 100 mls of serum.

Removing T and T, from normal serum or serum containing elevated TBG levels, such as by the methods outlined below, results in a serum judged hypothyroid both by methods which measure T concentration and T uptake.

Method 1. Add Item No. 2 to 2400 ml of Item No. l in a 4 liter Erlenmeyer flask (about 20% carbon weight to volume of serum).

2. Swirl gently at room temperature until the charcoal particles are dispersed in the liquid.

3. Place the mixture at 4C. Stir very gently for about 24 hrs.

4. At the end of 24 hrs., centrifuge mixture at high speed in a refrigerated centrifuge (34,800 X g).

5. Decant the supernatant and centrifuge it again as in step 4.

6. After the second centrifugation, again decant the supernatant and filter it by vacuum through a millipore filter.

7. Pour the filtrate (I200 ml) into a suitable glass tray for lyophilization and lyophilize.

8. Transfer the lyophilized material to a 2 liter Erlenmeyer flask and add Item No. 3.

9. Let the mixture stand for 30 minutes, then aid the dissolving process by swirling gently.

10. Add 300 ml of Item No. l to the serum mixture ob tained in step 9.

11. Dispense 2.14 cc into a vial that will hold a 2 ml fill and lyophilize.

Vials are reconstituted with 2.0 ml deionized distilled water. This serum is used for a hypothyroid control in conjunction with all thyroid function tests, i.e., the resulting serum control should have a T uptake level less than 25% by the Triosorb method and a T concentration less than 5.3 mg/100 ml by the Tetrasorb method.

Modifications in Example 1 are of course possible. Centrifugation (steps 4 and 5) may be eliminated; the millipore filter (step 6) may, of course, be an Ertel apparatus; the serum may be human or animal; the requirement to dispense the mixtures (step 11) may, of course, be modified or done away with completely; the time of mixing (step 3) may be shortened or lengthened over a range of 3-30 hours; the percentage of carbon added may range from 5-20 percent, with a range of about to about 20 percent being preferred.

This procedure removes over 99% of the T and T from the starting serum, effectively producing a T and T -free serum, while not significantly affecting the total protein concentration, pH, or T binding capacity of the serum.

The following example outlines the method used to remove T and T. from serum containing elevated TBG levels. Modifications of this example similar to those outlined for Example 1 are also possible and encompassed by this invention.

3. Place mixture at 4C and stir very gently for about 24 hours.

4. At the end of 24 hours, centrifuge mixture at high speed in a refrigerated centrifuge (34,800 Xg).

5. Decant the supernatent and centrifuge it again as in Step 4.

6. After second centrifugation, again decant the supernatent and filter it by vacuum through a sintered glass filter.

7. Add 300 ml of Item 3 to 600 m1 of serum obtained from Step 6. Swirl gently to mix.

8. Dispense 2.14 cc in a vial that will hold a 2 ml fill and lyophilize.

Vials are reconstituted with 2.0 ml deionized distilled water. This serum is used for a hypothyroid control serum in conjunction with all thyroid function tests.

The fill solution after reconstitution will contain about 7 percent protein. The resulting serum control should have a T uptake level less than 25% by the Triosorb Method and a T concentration less than 5.3ug% by the Tetrasorb method. The serum used in this example may be obtained, for example, from pregnant women or women on estrogen therapy.

A series of four separate experiments were carried out to show the effect on sera when treated as to Examples l and 2. The resultant sera were assayed by following the well recognized Triosorb and Tetrasorb protocols. The results of these experiments (Table I) show that serum treated according to the methods of this invention, may be used as a hypothyroid control serum EXAMPLE 2 for tests being carried out in clinical laboratories.

TABLE I Experiment Sera /1T;, Uptake T mcg 71 I Pooled Normal Human Serum (NHS) 27.1 7.4

Pooled Normal Human Serum after Example 1 treatment (HCS)* 15.1 0.8

II Pooled Normal Human Serum (NHS) 31.9 3.6

Pooled Normal Human Serum after Example 1 treatment (HCS)* 18.3 0.5 2 HCS: 1 NHS 21.2 3.3

m Pooled Normal Beef Serum (was) 29.3 11.4

Pooled Normal Beet" Serum after Example 1 treatment (HBS) 17.8 0.4 2 H88 1 NBS 19.3 24

IV Pooled Serum from Pregnant Women 17.6 13.0

Pooled Serum from Pregnant Women after Example 2 Treatment 13.6 0.3

This is the serum obtained after step 9 of Example I This is the serum obtained after step 10 of Example 1 Quantity for 900 ml Ingredients (420 vials) 1. Serum containing a high thyroxine 1100 ml binding globulin concentration 2. Neutral Decolorizing Carbon, 220 g pharmaceutical grade 3. Fresh. normal serum 300 m1 Method 1. Add Item 2 to Item 1 in a 2 liter Erlenmeyer flask (about 20% carbon weight to volume of serum).

2. Swirl gently at room temperature for one minute so that charcoal particles are dispersed in liquid.

The results of experiment I show that the pooled normal serum was euthyroid by both tests. After treatment the serum was hypothyroid for both T uptake and T concentration.

The results for experiment 11 show that the hypothyroid values may be altered upwardly when before treatment sera and after treatment sera are intermixed, as in this experiment in a 2 to 1 ratio.

ous ratiosof normal beef serum and the prepared hypothyroid beef serum.

Experiment IV shows the expected hypothyroid T uptake and euthyroid T concentration for pooled serum collected from pregnant women. After the treatment outlined in Example 2, however, the serum becomes hypothyroid for both T uptake and T concentration.

We claim;

1'. A method for obtaining a hypothyroid serum control comprising the following steps:

A. adding neutral, decolorizing carbon to blood serum in an amount of about 5-20 percent based upon weight of carbon to volume of serum;

B. mixing the above mixture for about 24 hours at a temperature of about 4C;

C. twice centrifuging the resulting slurry at 34,800 xg and at a temperature of about 4C;

D. filtering the resultant supernatant;

E. lyophilizing the supernatant" so obtained.

2. The method of claim 1 which includes adding untreated normal serum in an amount of about to about 35 percent of the serum volume of step A to the filtered supernatant of step D.

3. The method of claim 1 wherein the serum is animal blood serum.

4. The method of claim 1' wherein the serum is human blood serum.

5. The method of claim I wherein the serum is beef blood serum.

6. The method of claim 4 wherein the serum is T.,- euthyroid, hypothyroid, or hyperthyroid and T euthyroid or hypothyroid.

7. The method of claim 6 wherein the serum is T hypothyroid.

8. The method of claim 6 wherein the serum is T euthyroid and T -euthyroid.

9. A control serum judged hypothyroid by methods which measure T concentration and T uptake comprising blood serum which has been treated by the method according to claim 1.

10. In clinical chemistry procedures for measuring a persons serum triiodothyronine uptake level and thyroxine concentration, the improvement of which comprises the use of a single control serum standard which is a hypothyroid reference control serum for both triiodothyronine uptake level and thyroxine concentration tests.

11. The improvement of claim 10 in which the hypothyroid reference control serum preparation comprises the following steps:

A. adding neutral, decolorizing carbon to blood serum in an amount of about 5-20 percent based upon weight of carbon to volume of serum;

B. mixing the above mixture at a temperature of about 4C;

C. twice centrifuging the resulting slurry at 34,800 g and at a temperature of about 4C;

D. filtering the resultant supernatant;

E. lyophilizing the supernatant so obtained. 

1. A METHOD FOR OBTAINING A HYPOTHYROID SERUM CONTROL COMPRISING THE FOLLOWING STEPS: A. ADDING NEUTRA, DECOLORIZING CARBON TO BLOOD SERUM IN AN AMOUNT OF ABOUT 5-20 PERCENT BASED UPON WEIGHT OF CARBON TO VOLUME OF SERUM; B. MIXING THE ABOVE MIXTURE FOR ABOUT 24 HOURS AT A TEMPERATURE OF ABOUT 4*C; C. TWICE CENTRIFUGING THE RESULTING SLURRY AT 34,800 XG AND AT A TEMPERATURE OF ABOUT 4*C; D. FILTERING THE RESULTANT SUPERNATANT; E. LYOPHILIZING THE SUPERNATANT SO OBTAINED.
 2. The method of claim 1 which includes adding untreated normal serum in an amount of about 10 to about 35 percent of the serum volume of step A to the filtered supernatant of step D.
 3. The method of claim 1 wherein the serum is animal blood serum.
 4. The method of claim 1 wherein the serum is human blood serum.
 5. The method of claim 1 wherein the serum is beef blood serum.
 6. The method of claim 4 wherein the serum is T4-euthyroid, hypothyroid, or hyperthyroid and T3-euthyroid or hypothyroid.
 7. The method of claim 6 wherein the serum is T3-hypothyroid.
 8. The method of claim 6 wherein the serum is T4-euthyroid and T3-euthyroid.
 9. A control serum judged hypothyroid by methods which measure T4 concentration and T3 uptake comprising blood serum which has been treated by the method according to claim
 1. 10. In clinical chemistry procedures for measuring a person''s serum triiodothyronine uptake level and thyroxine concentration, the Improvement of which comprises the use of a single control serum standard which is a hypothyroid reference control serum for both triiodothyronine uptake level and thyroxine concentration tests.
 11. The improvement of claim 10 in which the hypothyroid reference control serum preparation comprises the following steps: A. adding neutral, decolorizing carbon to blood serum in an amount of about 5-20 percent based upon weight of carbon to volume of serum; B. mixing the above mixture at a temperature of about 4*C; C. twice centrifuging the resulting slurry at 34,800 g and at a temperature of about 4*C; D. filtering the resultant supernatant; E. lyophilizing the supernatant so obtained. 